Quantitative PCR assay for the detection of Aedes vigilax in mosquito trap collections containing large numbers of morphologically similar species and phylogenetic analysis of specimens collected in Victoria, Australia

Background Aedes vigilax is one of the most significant arbovirus vector and pest species in Australia’s coastal regions. Occurring in multiple countries, this mosquito species occurs as a species complex which has been separated into three clades with two detected in Australia. Until recently, Ae. vigilax has largely been absent from Victoria, only occasionally caught over the years, with no reported detections from 2010 to 2016. Complicating the detection of Ae. vigilax is the shared sympatric distribution to the morphologically similar Ae. camptorhynchus , which can exceed 10,000 mosquitoes in a single trap night in Victoria. Currently, there are no molecular assays available for the detection of Ae. vigilax . We aim to develop a quantitative PCR (qPCR) for the detection of Ae. vigilax , with the specificity and sensitivity of this assay assessed as well as a method to process whole mosquito traps. Methods Trapping was performed during the 2017–2020 mosquito season in Victoria in two coastal areas across these 3 consecutive years. A qPCR assay was designed to allow rapid identification of Ae. vigilax as well as a whole mosquito trap homogenizing and processing methodology. Phylogenetic analysis was performed to determine which clade Ae. vigilax from Victoria was closest to. Results Aedes vigilax was successfully detected each year across two coastal areas of Victoria, confirming the presence of this species. The qPCR assay was proven to be sensitive and specific to Ae. vigilax , with trap sizes up to 1000 mosquitoes showing no inhibition in detection sensitivity. Phylogenetic analysis revealed that Ae. vigilax from Victoria is associated with clade III, showing high sequence similarity to those previously collected in New South Wales, Queensland and Western Australia. Conclusions Aedes vigilax is a significant vector species that shares an overlapping distribution to the morphologically similar Ae. camptorhynchus , making detection difficult. Here, we have outlined the implementation of a specific and sensitive molecular screening assay coupled with a method to process samples for detection of Ae. vigilax in collections with large numbers of non-target species. Graphical abstract